Small-molecule Hsp90 inhibitors

ABSTRACT

The present disclosure provides inhibitors of Hsp90 and methods of making and using the same. Such compounds are useful as radioimaging ligands and for the treatment of cancer and other conditions where cell growth or maintenance depend on Hsp90 activity.

This application is a continuation of application Ser. No. 11/814,506,filed Jul. 23, 2007, now U.S. Pat. No. 7,834,181, which is a nationalstage of application serial no. PCT/US2006/003676, filed Feb. 1, 2006,which claims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalPatent Application No. 60/649,322 filed Feb. 1, 2005, the contents ofall of which are incorporated herein by reference.

FIELD OF THE INVENTION

This application relates to adenine derivatives effective as inhibitorsof Hsp90, and to the use of such molecules in therapeutic applications.The molecules of the invention have a 2′,4′,5′-substitution patterns onaryl substituted adenine rings. The molecules of the invention areuseful in therapeutic application and as radioimaging ligands.

BACKGROUND OF THE INVENTION

The chaperone heat shock protein 90 (Hsp90) is an emerging target incancer treatment due to its important roles in maintainingtransformation and in increasing the survival and growth potential ofcancer cells.¹ Hsp90 function is regulated by a pocket in the N-terminalregion of the protein that binds and hydrolyzes ATP.^(1a) Occupancy ofthis pocket by high affinity ligands prevents the dissociation of Hsp90client proteins from the chaperone complex and as a consequence, thetrapped proteins do not achieve their mature functional conformation andare degraded by the proteasome. Protein clients of Hsp90 are mostlykinases, steroid receptors and transcriptional factors involved indriving multistep-malignancy and in addition, mutated oncogenic proteinsrequired for the transformed phenotype.³ Examples include Her2, Raf-1,Akt, Cdk4, cMet, mutant p53, ER, AR, mutant BRaf, Bcr-Abl, Flt-3, Polo-1kinase, HIF-1 alpha and hTERT.^(1c-e) Degradation of these proteins byHsp90 inhibitors leads to cell-specific growth arrest and apoptosis incancer cells in culture, and to tumor growth inhibition or regression inanimal models. One such inhibitor,17-allyl-amino-desmethoxy-geldanamycin (17AAG,

FIG. 1A) has entered clinical trials in cancer patients in the US and UKand has shown early evidence of therapeutic activity when administeredalone or in combination with docetaxel^(.2) Despite these earlypromising results, 17AAG has several potential limitations. Mostprominent are its limited solubility and cumbersome formulation. It alsoexhibits dose and schedule dependent liver toxicity believed to becaused by the benzoquinone functionality.^(2a) Radicicol (RD, FIG. 1B) astructurally unrelated natural product, has biological activity similarto that of 17AAG but is not hepatotoxic,³ yet no derivative of thisclass has made it into clinic.

Making use of the peculiar bent shape of Hsp90 inhibitors and ofexistent Hsp90 crystal data, purine-scaffold derivatives with Hsp90inhibitory activities have been designed.⁴ The first synthesizedderivative of this class, PU3 (FIG. 1C), bound Hsp90 with moderateaffinity and elicited cellular effects that mimic 17AAG addition.⁵Preliminary efforts focused at improving the potency of this agent havemostly focused on modifying the left side adenine of the scaffold (FIG.2) and have led to the synthesis of several compounds with improvedactivity in both biochemical and cellular assays.⁶ One such compound,PU24FC1 (FIG. 1D) is a potent and selective inhibitor of tumor Hsp90 andexhibits anti-tumor activities in both in vitro and in vivo models ofcancer.⁷ Other purine-scaffold compounds with higher potency overPU24FC1 in in vitro models of cancer have subsequently beendisclosed.^(8,9) Although a significant number of derivatives has beencreated by these combined efforts, the nature and position ofsubstituents on the right side aryl moiety (X₁ and X₂ in FIG. 2) has notbeen sufficiently investigated. (See also, PCT Patent Publications Nos.WO02/36705 and WO03/037860, which are incorporated herein by reference.)

The present invention provides a class of Hsp90 inhibitors with enhancedactivity as compared to previously known compounds and a class ofinhibitors with differential selectivity and activity for Rb normalversus Rb defective cells

SUMMARY OF THE INVENTION

In accordance with the present invention, Hsp90 inhibitors are providedhaving the formula:

wherein Y is C, N or O,

R is hydrogen, a C₁ to C₁₀ alkyl, alkenyl, alkynyl, or an alkoxyalkylgroup, optionally including heteroatoms such as N or O, or a targetingmoiety connected to N9 via a linker,

X₄ is hydrogen or halogen, for example F or Cl, or Br;

X₃ is CH₂, CF_(2,) S, SO, SO₂, O, NH, or NR², wherein R² is alkyl; and

X₂ is halogen, alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl,pyrollyl, optionally substituted aryloxy, alkylamino, dialkylamino,carbamyl, amido, alkylamido dialkylamido, acylamino, alkylsulfonylamido,trihalomethoxy, trihalocarbon, thioalkyl, SO₂₋alkyl, COO-alkyl, NH₂, OH,CN, SO₂X₅, NO₂, NO, C═S R₂ NSO₂X_(5,), C═OR_(2,) where X₅ is F, NH2,alkyl or H, and R₂ is alkyl, NH₂, NH-alkyl or O-alkyl; and

X₁ represents two substituents, which may be the same or different,disposed in the 4′ and 5′ positions on the aryl group, wherein X₁ isselected from halogen, alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl,pyrollyl, optionally substituted aryloxy, alkylamino, dialkylamino,carbamyl, amido, alkylamido dialkylamido, acylamino, alkylsulfonylamido,trihalomethoxy, trihalocarbon, thioalkyl, SO₂₋alkyl, COO-alkyl, NH_(2,)OH, CN, SO₂X₅, NO₂, NO, C═SR₂ NSO₂X_(5,), C═OR_(2,) where X₅ is F, NH2,alkyl or H, and R₂ is alkyl, NH₂, NH-alkyl or O-alkyl, C₁ to C₆ alkyl oralkoxy; or wherein X₁ has the formula —O—(CH₂)_(n)—O—, wherein n is aninteger from 0 to 2, and one of the oxygens is bonded at the 5′-positionand the other at the 4′-position of the aryl ring.

The Hsp90 inhibitors can be used for therapeutic application in thetreatment of cancer and other conditions where the cells depend on hsp90activity for cell growth or maintenance. Radiolabeled Hsp90 inhibitorsof the invention are useful as radiotracers for imaging tumors thatexpress Hsp90.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B shows the structure of prior art Hsp90 inhibitors 17-AAGand radicicol.

FIGS. 1C and 1D shows the structure of prior art purine-scaffolded Hsp90inhibitor PU3 and PU24FC1.

FIG. 2 shows a general structure of a purine-scaffold Hsp90 inhibitor.

FIG. 3 A-Q shows the structures of various purine-scaffolded Hsp90inhibitors in accordance with the present invention.

FIG. 4 shows the structure of an exemplary Hsp90 inhibitor connected toa targeting moiety via a connector.

FIGS. 5A-D shows synthetic schemes for making compositions in accordancewith the invention.

FIGS. 6A and B compare the effectiveness of PU24FC1 and a compound ofthe invention against various small cell lung cancer cells lines.

FIGS. 7A-D show experimental results for compounds in accordance withthe invention, as compared to PU24FC1.

FIG. 8 shows inhibition of cell growth and induction of cell death inSCLC cells highly resistant to conventional chemotherapy using Example 9in accordance with the invention.

FIGS. 9A-D show a comparison of Example 9 in accordance with theinvention and prior art compounds for efficacy against small cell lungtumors.

FIGS. 10A and B show pharmacodynamic effects of Compound 6 whenadministered to MDA-MB bearing mice.

FIGS. 11 A and B show enhancement of radiation sensitivity in gliomacells after pretreatment with to Compounds 9 or 8, respectively.

FIG. 12 shows example displacement curves for [¹³¹I]-Compound 9 and[¹³¹I]-Compound 9.

FIG. 13 shows saturation curves [¹³¹I]-Compound 8 and [¹³¹ I]-Compound9.

FIG. 14 shows the ratio of [¹³¹I]-Compound 8 and [¹³¹I]-Compound 9 intumor and muscle and tumor and blood 4 and 24 hours afteradministration.

DETAILED DESCRIPTION OF THE INVENTION Compositions of the Invention

The present application provides small molecule Hsp90 inhibitors thatare purine-scaffold derivatives of adenine with the general structure:

wherein Y is C, N or O,

R is hydrogen, a C₁ to C₁₀ alkyl, alkenyl, alkynyl, or an alkoxyalkylgroup, optionally including heteroatoms such as N or O, or a targetingmoiety connected to N9 via a linker,

X₄ is hydrogen or halogen, for example F or Cl, or Br;

X₃ is CH₂, CF_(2,) S, SO, SO₂, O, NH, or NR², wherein R² is alkyl; and

X₂ is halogen, alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl,pyrollyl, optionally substituted aryloxy, alkylamino, dialkylamino,carbamyl, amido, alkylamido acylamino, alkylsulfonylamido,trihalomethoxy, trihalocarbon, thioalkyl, SO₂₋alkyl, COO-alkyl, NH_(2,)OH, CN, SO₂X₅, NO₂, NO, C═S R_(2,) NSO₂X_(5,), C═OR_(2,) where X₅ is F,NH2, alkyl or H, and R₂ is alkyl, NH₂, NH-alkyl or O-alkyl; and

X₁ represents two substituents, which may be the same or different,disposed in the 4′ and 5′ positions on the aryl group, wherein X₁ isselected from halogen, alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl,pyrollyl, optionally substituted aryloxy, alkylamino, dialkylamino,carbamyl, amido, alkylamido dialkylamido, acylamino, alkylsulfonylamido,trihalomethoxy, trihalocarbon, thioalkyl, SO₂₋alkyl, COO-alkyl, NH_(2,)OH, CN, SO₂X₅, NO₂, NO, C═SR₂ NSO₂X_(5,), C═OR_(2,) where X₅ is F, NH2,alkyl or H, and R₂ is alkyl, NH₂, NH-alkyl or O-alkyl, C₁ to C₆ alkyl oralkoxy; or wherein X₁ has the formula —O—(CH₂)_(n)—O—, wherein n is aninteger from 0 to 2, and one of the oxygens is bonded at the 5′-positionand the other at the 4′-position of the aryl ring.

The right-side aryl group may be phenyl as shown, or may include one ormore heteroatoms. For example, the right-side aryl group may be anitrogen-containing aromatic heterocycle such as pyrimidine.

In specific preferred embodiments of the composition of the invention,the right side aryl group X₁ has the formula —O—(CH₂)_(n)—O—, wherein nis an integer from 10 to 2, preferably 1 or 2, and one of the oxygens isbonded at the 5′-position of the aryl ring and the other at the 4′position. In other specific embodiments of the invention, thesubstituents X₁ comprise alkoxy substituents, for example methoxy orethoxy, at the 4′ and 5′-positions of the aryl ring.

In specific embodiments of the invention, the substituent X₂ is ahalogen.

In specific embodiments of the invention, the linker X₃ is S. In otherspecific embodiments of the invention, the linker X₃ is CH₂.

In specific embodiments of the invention, R is a pent-4-ynylsubstituent. In other specific embodiments of the invention, R containsa heteroatom, for example nitrogen. A preferred R group that increasesthe solubility of the compound relative to an otherwise identicalcompound in which R is H or pent-4-ynyl is —(CH₂)_(m)—N—R₁₀R₁₁R₁₂, wherem is 2 or 3 and where R₁₀₋₁₂ are independently selected from hydrogen,methyl, ethyl, ethene, ethyne, propyl, isopropyl, isobutyl, ethoxy,cyclopentyl, an alkyl group forming a 3 or 6-membered ring including theN, or a secondary or tertiary amine forming a 6-membered ring with thenitrogen. In specific examples, R₁₀ and R₁₁ are both methyl, or one ofR₁₀ and R₁₁ is methyl and the other is ethyne.

FIG. 3A shows exemplary structures in accordance with the presentinvention. The structures are based on one of two right side arylsubstitution patterns in which the 4′ and 5-substituents are either abridging substituent or dihydroxy/alkoxy. The left side adenine hasvarying substituents (Cl, F or H) at the 2-position, and choices for theN9 substituent R are listed. FIGS. 3 B-Q shows additional options forspecific structures in accordance with the invention.

Synthesis of Compositions in Accordance with the Invention

Synthesis of sulfur linker derivatives can be achieved using theprocedures as outlined in Scheme 1 (FIG. 5A). Formation of a sulfur linkbetween adenine and the phenyl ring (X3=S) may be obtained either bynucleophilic attack of the arylthiolate anion on 8-bromoadenine (Step a,Scheme 1) or by the copper catalyzed coupling of aryliodides withmercaptoadenine (Step b, Scheme 1). Our developed method for theformation of 8-arylsulfanyl adenine derivatives (5) from8-mercaptoadenine (3) and aryl iodides (4) uses CuI/neocuproine ascatalyst and NaOt-Bu/DMF as the base/solvent combination.^(11c) Thereaction occurs in anhydrous DMF at 110° C. under nitrogen to generatethe products in good yields. If bromine or chlorine is present on thearyl moiety, the coupling requires milder basic condition and utilizesNa₃PO₄ or K₂CO₃ instead of NaOt-Bu. Although less attractive due tothiophenols' limited commercial availability, stench and tendency toquickly oxidize, coupling of 8-bromoadenine (1) with thiophenols 2 inthe presence of a base will also be used. When not commerciallyavailable, thiophenols can be generated by a modified Leuckartthiophenol reaction starting from the corresponding aryl amine. In afirst step, aryl amines will be converted via the aryl diazonium salt toaryl xanthates which afford thiophenols on reduction with LiAlH4 or onwarming with a base solution. The 8-arylsulfanyl adenines 5 obtained inthese coupling reactions will be further alkylated at the position 9-Nwith pent-4-ynyl or the corresponding -bromoalkylalcohols. Theirintroduction will be carried out using a Mitsunobu type reaction^(13b)between the alcohol and the respective 8-arylsulfanyl adenines 5 intoluene/CH₂Cl₂ to result in the corresponding 9-N-alkyl-8-arylsulfanyladenines 6. Formation of the 3-N and 7-N isomers is likely to beobserved (˜0 to 30%), however, these byproducts will be removed bycolumn chromatography (differences in Rf are considerable). Treatment of5 with base and an alkylating agent tends to result in higher percentageof 3- and 7-N isomers compared to alcohol treatment under the Mitsunobuconditions. Heating these bromines with amines in DMF will generate thedesired products. Phosphate salts will be further made for in vivoadministration of selected compounds to improve their water solubility.

Synthesis of methylene linker derivatives can be accomplished using theprecoures outlined in Scheme 2 (FIG. 5B). If the linker is methylene(X3=CH₂), synthesis will commence with coupling of the commerciallyavailable 2,4,5,6-tetraminopyrimidine sulfate (9) with the acid fluoride(8) of the corresponding carboxylic acids. We have previously determinedthis to be the only coupling method that in our hands gives the productin high yields. Acid fluorides are generated by treating thecorresponding carboxylic acids with cyanuric fluoride and pyridine inCH₂Cl₂. Following a quick water wash, the resulted acid fluorides areused in the next step without further purification. To make thenecessary 2-chloro, bromo or iodo, 4,5-derivatized phenyl acetic acidswe have determined optimal reaction conditions. We have identified thattreatment of 4,5-derivatized phenyl acetic acids with ICl/AcOH; NBS; orHCl/t-BuOOH gives solely ortho-iodinated; brominated; or chlorinatedproduct, respectively in high yields. The amides resulted from thepyrimidine-acid fluoride couplings will be cyclized by heating inalcoholic NaOMe. Transformation of the C2-amino group to fluorine(X₄═NH₂ to F) will be conducted by diazotization-fluorodediazoniation ofthe amino derivative in HF/pyridine in the presence of NaNO₂ to yieldthe corresponding adenine derivatives 10. This reaction givessignificantly higher yields over the method using HBF₄/iso-amyl nitrite,likely to be due at least in part to the more anhydrous nature of thissolvent system which reduces the proportion of hydrolysis. Furtheralkylation will be conducted using the reaction sequence mentioned inScheme 1 to result in derivatives 12.

Several derivatives were prepared by the introduction of fluorine atposition C2 of the adenine moiety (Scheme 3, FIG. 5C). We andothers^(6,8,9) have previously determined that fluorine in this positionin general increased the solubility and/or potency of the resultingpurines. For the preparation of such derivatives, synthesis commencedwith the condensation of the commercially available2,4,5,6-tetraminopyrimidine sulfate (14) with carbon disulfide in arefluxing solution of NaHCO₃ in aqueous EtOH.¹⁷ The resulting2-amino-8-mercaptoadenine (15) was further coupled with aryl iodides inthe presence of NaOt-Bu and CuI in ethylene glycol11b to give2-amino-8-arylsulfanyl adenines 16. The reaction did not proceed usingthe method published by He et al^(11c) as 15 did not dissolve in DMF ormost organic solvents for that matter. Unsaturated chains at position9-N were introduced at this stage to result in the2-amino-9-N-alkyl-8-arylsulfanyl adenines 17. The reaction required theuse of K₂CO₃ or Cs₂CO₃ and an alkylating agent in DMF due to the poorsolubility of 16 in the Mitsunobu reaction solvent. If further,introduction of chlorine at X₂ on the aryl moiety was desired, compounds17 were subjected to HCl/t-BuOOH to result preferentially inorto-chlorinated compounds (18). Transformation of the C2-amino group tofluorine (X₄═NH₂ to F) was conducted bydiazotization-fluorodediazon-iation of the amino derivative inBF/pyridine in the presence of NaNO₂ ^(6,18) to yield the corresponding2-fluoro-9-N-alkyl-8-arylsulfanyl adenine derivatives 21. This reactiongave significantly higher yields over the previously published method⁹using HBF₄/iso-amyl nitrite, likely to be due at least in part to themore anhydrous nature of this solvent system which reduces theproportion of hydrolysis. Synthesis of derivatives containing both anunsaturated chain at 9-N (i.e. pent-4-ynyl) and fluorine at the C₂position of the adenine moiety (X₄═F) required a different strategy toavoid fluorine addition to the triple bond. Compound 16 was firstsubjected to introduction of fluorine at C₂ to result in thecorresponding 2-fluoro-8-arylsulfanyl adenine 19. Surprisingly,conducting the reaction at room temperature resulted in addition offluorine at both C2 and C6 of the purine moiety. This problem wasaverted by lowering the reaction temperature to −40° C. Addition offluorine at position C₂ significantly increased the solubility of thesepurines in organic solvents. Thus, further alkylation could be easilyconducted using the Mitsunobu reaction in toluene:CH2Cl2 to result inderivatives 20. Introduction of chlorine (X₂═Cl), again with HCl/t-BuOOHled to formation of 21. Alternatively, synthesis of2-fluoro-9-N-alkyl-8-arylsulfanyl adenines 20 was started with thecommercially available 2-fluoroadenine (22) (Scheme 3). This reagent wasfirst 9-N alkylated by Cs₂CO₃/tosylate treatment followed by8-bromination with N-bromosuccinimide (NBS)19 to result in2-fluoro-9-N-alkyl-8-bromo adenine 23. Its coupling with thiophenolsgenerated the 2-fluoro-9-N-alkyl-8-arylsulfanyl adenines 20.

Syntheses of 8-arylsulfoxyl adenine derivatives 27 and 8-arylsulfonyladenine derivatives 28 (Scheme 4, FIG. 5D) were previously reported byLlauger et al.²⁰ Briefly, synthesis started with the preparation ofC₆—NH2 triphenylphosphine protected²¹ 9-N-alkyl-8-arylsulfanyl adeninederivatives 24 in a two-step (alkylation-protection) one-pot reactionfrom the corresponding 8-arylsulfanyl adenines 10 using the Mitsunobuconditions13b followed by addition of an excess of PPh3 anddi-tert-butyl azodicarboxylate (DBAD). Oxidation of 24 with OXONE® inthe presence of alumina 22 allowed for monitoring the reaction to eithersulfoxide (25) or sulfone (26). Deprotection of the C6-NH₂triphenylphosphine group was conveniently conducted in refluxingAcOH/EtOH²¹ to result in good yields in the corresponding 8-arylsulfoxyladenine derivatives 27 and 8-arylsulfonyl adenine derivatives 28. Thelater could be chlorinated at C′₂ (X₂═CI) by HCl/t-BuOOH treatment toresult in derivatives 29.

Using these methods, the compositions described in this application wereprepared. These compositions include various compositions in accordancewith the invention, as well as comparative examples.

Biological Testing

Compounds synthesized above were tested in a biochemical assay, and alsoin cellular assays that probe for cellular fingerprints of Hsp90inhibition.¹ The biochemical assay tests competitive binding ofcompounds to recombinant Hsp90α protein and also Hsp90 found in cellspecific complexes, and uses a fluorescence polarization method.^(7,23)When using cell lysates instead of recombinant protein, the assaymeasures binding to average Hsp90 population found in cell specificcomplexes.⁷ The cellular assays measure two specific biological effectsobserved upon addition of known Hsp90 inhibitors to cancer cells: (a)degradation of the tyrosine kinase Her224 and (b) mitotic block inRb-defective cells.²⁵ Overexpression of the receptor tyrosine kinaseHer2 in SKBr3 breast cancer cells leads to Akt activation which in turnpromotes cell survival. Hsp90 uniquely stabilizes Her2 via interactionwith its kinase domain and an Hsp90 inhibitor induces Her2 degradationby disrupting the Her2/Hsp90 association.²⁴ We have previously reporteda fast microtiter immunoassay able of quantifying cellular levels ofHer2 following drug treatments.²⁶ This assay is used here todifferentiate the Her2-degradative

potential of the above synthesized purines. Hsp90 inhibitors are alsoknown to cause cells lacking

functional RB to progress normally through G1 and arrest in mitosis.²⁵Thus, another assay used here to test cellular Hsp90 inhibition relieson assessing the anti-mitotic potential of synthesized purines. Theassay is a microtiter immunoassay and uses an antibody against amitotically phosphorylated form of nucleolin to detect cells inmitosis.²⁷ This antibody (Tg-3), originally described as a marker ofAlzheimer's disease, is highly specific for mitotic cells, Tg-3immunofluorescence being >50-fold more intense in mitotic cells than ininterphase cells.²⁸ In addition, the cytotoxicity of these agentsagainst SKBr3 breast cancer cells was determined. Aselected number of most active purines were also tested for possibletoxicity against a normal cell line, renal proximal tubular epithelialcells (RPTEC).⁷

Table 1 shows compounds that were tested for biological activity. Thesecompounds are identified by an example number in the table, and arereferred to herein by that number as Example _(——————) or Compound_(——————). In these compounds, Y₁ and Y₂ are both CH. Table 2 showsresults for EC50, Hsp90 α, IC₅₀ for Her2 degradation, and IC₅₀ forgrowth inhibition in SKBr3 breast cancer cells. All values in Table 2are in μM and represent an average of 3 measurements. As can be seen,the compositions tested all show substantial activity, and in many casesactivity at nanomolar concentrations.

Several active derivatives were tested for specificity towardstransformed cells (Table 3). Binding affinities of selected compoundsfor average population Hsp90 complexes found in normal tissues (brain,lung and heart) and in addition, their cytotoxicities against RPTECnormal cells were determined. Compounds were found to bind Hsp90 fromnormal tissues with 2- to 3-log weaker affinities when compared to Hsp90from SKBr3 cells. This specificity translated into 5 to 100-foldselectivity (column 10, Table 3) in inhibiting the growth of transformedcells compared to cultured normal epithelial cells (RPTEC tested). Nocell death was observed in the purine-scaffold treated RPTEC cells evenat the highest tested concentrations. Selectivity was also observedbetween SKBr3 cells and MRC5 normal lung fibroblasts for compounds 9 and10.

TABLE 1 Example X1 X2 X3 X4 R 1 —OCH₂O— Br S H pent-4-ynyl 2 Cl, Cl Cl SH pent-4-ynyl 3 4-Cl, 5-methoxy Cl S H 2-isopropoxy-ethyl 4 Cl, Cl Cl SH H 5 —OCH₂O— Br S H 3-isopropylamino- propyl 6 —OCH₂O— Br CH₂ Fpent-4-ynyl 7 —OCH₂O— Cl CH₂ F pent-4-ynyl 8 —OCH₂O— I CH₂ F3-isopropylamino- propyl 9 —OCH₂O— I S H 3-isopropylamino- propyl 10—OCH₂O— I S H pent-4-ynyl 11 —OCH₂O— I CH₂ F pent-4-ynyl 124,5-dimethoxy I CH₂ F pent-4-ynyl 13 4,5-dimethoxy Br CH₂ F pent-4-ynyl14 4,5-dimethoxy Cl CH₂ F pent-4-ynyl 15 —OCH₂O— I CH₂ H3-isopropylamino- propyl Comp 1 3′,4′,5′- Cl CH₂ F pent-4-ynyl (PU24FCl)trimethoxy Comp 2 3′,4′,5′- Cl S F pent-4-ynyl trimethoxy

TABLE 2 EC₅₀ Hsp90-α IC₅₀ Her2 IC₅₀ SKBr3 Anti- Example (μM) (μM) (μM)mitotic 1  0.03 ± 0.005  0.3 ± 0.05  0.2 ± 0.01 Yes (2exp) 0.0508 ±0.004  0.365 ± 0.045 0.300 ± 0.05  2 8.5 ± 0.1  58 ± 1.4 48.4 ± 2.5  No3 5.0 ± 1.1 58.3 ± 2.2  16.1 ± 0.7  No 4 15.4 ± 2.2  47.8 ± 3.2  36.0 ±0.2  Yes 5 0.0388 ± 0.003  0.205 ± 0.015 0.142 ± 0.022 Yes 6 0.0565 ±0.002  0.210 ± 0.01  0.215 ± 0.055 Yes 7 0.0772 ± 0.001  0.300 ± 0.0150.250 ± 0.030 Yes 8 0.0504 ± 0.004  0.080 ± 0.010 0.045 ± 0.006 Yes 90.0161 ± 0.001   0.05 ± 0.006  0.05 ± 0.005 Yes 10 0.010.8 ± 0.002  0.100 ± 0.010 0.090 ± 0.002 Yes 11 0.0223 ± 0.002  0.090 ± 0.01  0.090 ±0.03  Yes 12 >15 >50 >50 No 13 >15 >50 55.0 ± 2.3  No 14  4.6 ± 0.0230.0 ± 1.0  20.6 ± 6.7  No Comp 2 0.12 ± 0.3  1.3 ± 0.4 1.8 ± 0.2 Yes

TABLE 3 EC₅₀ EC₅₀ EC₅₀ EC₅₀ IC₅₀ Hsp90 Hsp90 Hsp90 Hsp90 Brain/ Lung/Heart/ IC₅₀ RPTEC/ MRC5 MRC5/ Exam Brain lung heart SKBr3 SKBr3 SKBr3SKBr3 RPTEC SKBr3 cells SKBr3 1 40.2 ± 25.3 14.7 ± 1.7  65.3 ± 9.9  0.02± 0.06 2000 735 3265 4.1 ± 0.9 20.5 6 ND  5.20 ± 0.210 13.30 ± 0.210.0388 ± 0.003 ND 134 343 ND ND 9 ND 2.40 ± 0.24 6.90 ± 0.15 0.0504 ±0.004  ND 48 140 ND ND >5 >50 10  ND 2.20 ± 0.40 6.00 ± 0.20 0.0161 ±0.001  ND 136 370 ND ND 1 20 Comp 2 26.7 ± 7.7  217 ± 50  53.1 ± 20.50.09 ± 0.03  298 2400 590 28.1 ± 2.2  15.6

A compound in accordance with the invention having the structure shownin FIG. 3B (Compound 1) and PU24FC1, (Comp 1, FIG. 1D) were tested foractivity against 7 different small cell lung cancer cell lines (NCI-H69,NCI-H146, NCI-H209, NCI-H187, NCI-N417, NCI-H510 and NCI-H256) obtainedfrom the American Type Culture Collection (Manassas, Va.). Bothcompounds exhibited binding to cellular Hsp90 as determined byhomogenous fluorescence polarization. However, the concentrationrequired of the compound of the invention was about 1 order of magnitudeless than for PU24FC1. (Table 4) Antiproliferative effects of the twocompounds were observed in the SCLC cell lines, and the percentage ofapoptotic cells were determined. As shown in FIG. 6A (PU24FC1) and B(Example 1), both compounds induced apoptosis in each of the SCLC cellslines, but the composition of the invention does so at a lowerconcentration. Both PU24FC1 and its higher potency derivative, Example1, inhibit the growth and cause significant cytotoxicity against allSCLC cell lines. At concentrations of Hsp90 inhibitors in excess of 4-12mM and 0.25-1 mM, respectively, 50-100% of the starting cell populationsare dead at 72 h or 96 h post-treatment. As shown in Table 4 and FIGS.6A and B, the 10 to 20-fold difference in binding for the two drugscorrelate well with the their anti-proliferative potencies, suggestingthat cell death in these cells is a direct result of Hsp90 inhibition.

TABLE 4 IC₅₀ IC₉₀ EC₅₀ IC₅₀ IC₉₀ EC₅₀ IC₅₀ EC₅₀ Comp 1 Comp 1 Comp 1Examp 1 Examp 1 Exampl 1 Comp 1/Ex 1 Comp 1/Ex 1 NCI-H526 4.2 ± 0.6  7.4± 0.1 0.229 ± 0.04 0.44 ± 0.04 0.83 ± 0.05 0.023 ± 0.02 9.5  9.6NCI-N417 3.5 ± 0.3  5.3 ± 0.4 0.592 ± 0.13 0.33 ± 0.03 0.61 ± 0.01 0.038± 0.01 10.6 15.4 NCI-H146 5.8 ± 0.2 10.4 ± 0.2 0.574 ± 0.22 0.27 ± 0.040.49 ± 0.02 0.049 ± 0.02 21.5 13.1 NCI-H187 6.5 ± 0.3 12.5 ± 0.5 ND 0.64± 0.02  1.2 ± 0.03 ND 8.3 ND NCI-H209 9.5 ± 0.5 11.6 ± 0.4 0.347 ± 0.060.50 ± 0.05 0.65 ± 0.04 0.021 ± 0.04 19.0 16.0 NCI-H510 10.7 ± 0.1  13.4± 0.6 ND 0.76 ± 0.02 0.90 ± 0.09 ND 14.1 ND NCI-H69 2.5 ± 0.2  4.0 ± 0.10.350 ± 0.1  0.17 ± 0.02 0.25 ± 0.03 0.019 ± 0.01 14.7 17.8

FIGS. 3B-E show the structure of four compounds (examples 1, 5, 6 and 7,respectively) in accordance with the invention made using the methods ofSchemes 1 and 2. Where the 9N-alkyl chain is pent-4-ynyl, 5 and 10 weredirectly alkylated with using the Mitsonobu reaction and thecorresponding alcohol. FIG. 7A-D shows test results for these compoundswhen compared to the prior art compound of FIG. 1D (Comp 1). FIG. 7Ashows results when exponentially growing SKBr3 cancer cells were seededinto 96-well plates and incubated in medium containing either of vehiclecontrol (DMSO) or the test compound (four replicate wells per assaycondition) at the above indicated concentrations for 72 h at 37° C. Theantiproliferative effects of test compounds were evaluated using theCellTiter-Glo® Luminescent Cell Viability Assay kit from PromegaCorporation. FIG. 7B shows results when exponentially growing SKBr3cancer cells were seeded into 96-well plates and incubated in mediumcontaining either of vehicle control (DMSO) or the test compound (fourreplicate wells per assay condition) at the above indicatedconcentrations for 72 h at 37° C. The antiproliferative effects of testcompounds were evaluated using the CellTiter-Glo® Luminescent CellViability Assay kit from Promega Corporation. FIG. 7B shows results whenexponentially growing SKBr3 cancer cells were seeded into 96-well platesand incubated in medium containing either of vehicle control (DMSO) orthe test compounds (four replicate wells per assay condition) at theabove indicated concentrations for 24 h at 37° C. The Her2 degradationpotential of PUs was evaluated using our Her2 blot procedure describedin PCT Patent Application Serial No. PCT/US04/21297, which isincorporated herein by reference. FIG. 7C shows results whenexponentially growing MDA-MB-468 cancer cells were seeded into 96-wellplates and incubated in medium containing either of vehicle control(DMSO) or the test compounds (four replicate wells per assay condition)at the above indicated concentrations for 24 h at 37° C. Theanti-mitotic potential of compounds was evaluated using our Tg3 blot.The resulted chemiluminescent signal was read with an ANALYST ADmicroplate reader. FIG. 7D shows the apoptotic inducing potential ofPUs, which was assessed using caspase-3,7 activation as read-out.MDA-MB-468 cells (plated at 8,000/well) were plated in 96-well platesand treated for 24 h with varying concentration of compounds. For thecaspase-3,7 assay, following treatment cells are lysed and permeabilizedwith our in-house developed buffer (10 mM HEPES pH. 7.5, 2 mM EDTA, 0.1%CHAPS, 0.1 mg/ml PMSF, COMPLETE Protease Inhibitor Mix) to make themaccessible to the caspase-3,7 substrate Z-DEVD-R110. This agent becomeshighly fluorescent upon cleavage by activated caspase-3,7 and subsequentrelease of rhodamine, thus the assay is a simple read-mix procedure. Theresulted fluorescence signal will be read using the SPECTRAMAX GEMINI XS(Molecular Devices) (ex.485, em.530). As is apparent from these figures,the four compositions of the invention all produced similar results toone another, but were active at significantly lower concentrations thanthe comparison compound.

The compounds of FIGS. 3F and 3G (Examples 8 and 9) are water solublewhen in the acid addition salt form (solubility>5 mM in PBS) and havelow nanomolar potency against cancer cells. The pharmacokinetic andpharmacodymanic profiles, as well as the anti-cancer activity ofCompound 9 were tested in a model of small cell lung cancer. Small celllung carcinoma (SCLC) is a highly malignant tumor accounting for about20% of all lung cancers. It is a disease that metastasises early andwidely, accounting for the extremely poor prognosis of this tumor.Whereas it often initially responds well to chemotherapy, relapses occuralmost without exception, and they are usually resistant to cytotoxictreatment. We have shown that compound of FIG. 3G (Example 9) retainsits activity in H69AR, a SCLC cell line resistant to adriamycin as wellas in SKI-Chen, a cell line derived at MSKCC from a patient who failedto respond to every conventional therapy. The H69AR line was establishedfrom NCI-H69 cells that were grown in the presence of increasingconcentrations of adriamycin (doxorubicin) over a total of 14 months.The cell line is cross-resistant to anthracycline analogues includingdaunomycin, epirubicin, menogaril, and mitoxantrone as well as toacivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids,vincristine and vinblastine, and expresses the multidrug resistanceprotein. Growth over 96 h was assessed. FIG. 8 shows a comparison ofresults for Compound 9 and PU24FC1. Values below 0% represent cell deathof the starting population. As shown, the compound of the invention iseffective at a concentration about 20 times lower.

In vivo, Example 9 exhibits the tumor retention profile manifested byour early micromolar compound of FIG. 1D (Comp 1) as shown in FIGS. 9Aand B. While Example 9 is rapidly cleared from plasma, with levelsundetectable after 6 h post-administration, it is retained in tumors atpharmacologic doses for more than 36 h. Such behavior translates inconsiderable downregulation for more than 36 h of Hsp90 client proteinsdriving transformation in this tumor. Proteins involved in growth andsurvival potential of the tumor, Raf-1 and Akt, are efficiently degradedor inactivated (pAkt) by one administered dose of Example 9. Inaddition, the agent induces significant apoptosis of the tumor asreflected by an increase in PARP cleavage. (FIG. 9C) This is the firstindication of apoptosis induced in vivo by an Hsp90 inhibitor.Concordantly, Example 9 efficiently inhibited the growth of this tumorin a manner comparable to 17AAG without toxicity to the host (FIG. 9D).

Mice xenografted with MDA-MB-468 human breast cancer tumors were treatedby intraperitoneal injection with Compound 5 at a dosage levels of 25,50, 75, 100 and 150 mg/kg. The concentration of Compound 5 in the tumor24 hours after administration was determined. As reflected in FIG. 10A,at all dosage levels a biologically active concentration of 300 nM wasobserved in the tumor, although it could not be detected in plasma.Compound 5 was further tested for its effects on the pharmacodynamicmarker Raf-1 kinase. Hsp90 stabilizes this kinase and maintains it inready-to-be-activated conformation. Inhibition of Hsp90 leads todisruption of the complex and further ubiquitinylation and degradationof Raf-1 by the proteasome. Thus, Raf-1 degradation in tumors is afunctional read-out of Hsp90 inhibition. FIG. 10B shows % control levelsof Raf-1 protein in mice to which Compound 5 was administered. As shown,substantial decrease in Raf-1 levels was observed at all dosage levels.In contrast, no change in expression of PI3 kinase, a protein unaffectedby Hsp90 inhibitors.

Observation of Selective Activity

Although the change of the linker structure between CH₂ and S did notresult in significant change in activity measured in the biologicalassays described above, this work has allowed the identification ofHsp90 inhibitors that demonstrate selective affinities for certainHsp90-client protein complexes. Compounds Example 1 and Comp 2 inducedHer2 degradation and inhibition of growth in SKBr3 cells, and alsoexhibited anti-mitotic activity in MDA-MB-468 cells, these eventsoccurring with similar potencies. However, among the moderate affinitybinders, derivatives were identified that degrade Her2 withcorresponding potencies but do not affect cell cycle distribution inRB-defective cells at similar concentrations. The Hsp90 client proteinof whose inactivation by Hsp90 inhibitors is responsible for the blockof these cells in mitosis is currently unknown. Due to their selectivityprofile, these derivatives may be useful pharmacological tools indissecting Hsp90-regulated processes.

A study comparing the activity of pairs of compounds differing only inthe nature of the linker indicated that in general, compounds with CH₂as the linker are antimitotic, while compounds with S as the linker arenot. Consistent with this observation, caspase 3,7 assays showed thatcompounds with a CH₂ linker induce apoptosis in Rb defective cells,while the S compound does not, This is indicative of a selectiveaffinity to hsp90 complexes in these cells. Both S and CH₂ compounds mayhave comparable affinity for hsp90 complexes that regulate cell growthand survival, regardless of Rb-type, while the S compounds are moreweakly bound to hsp90 complexes that regulater transition throughmitosis in Rb-defective cells. As a result of this selectivity, Scompounds are more beneficial in the treatment of diseases/conditionswhere apoptotis is not desired. This would include neurodegnerativediseases, ischemia, inflammation, HIV and nerve regeneration.

Compositions Coupled to Targeting/Labeling Moieties

The compounds of the invention may be coupled via N9 to a targetingmoiety selected to specifically bind to a protein, receptor or markerfound on a target population of cells. The targeting moiety may be ahormone, hormone analog, protein receptor- or marker-specific antibodyor any other ligand that specifically binds to a target of interest, andis selected on the basis of the identity of the target. Particulartargeting moieties bind to steroid receptors, including estrogen andandrogen and progesterone receptors, and transmembrane tyrosine kinases,src-related tyrosine kinases, raf kinases and PI-3 kinases. Specifictyrosine kinases include HER-2 receptors and other members of theepidermal growth factor (EGF) receptor family, and insulin andinsulin-like growth factor receptors. Examples of specific targetingmoieties include estrogen, estradiol, progestin, testoterone, tamoxifenand wortmannin. Targeting moieties may also be antibodies which bindspecifically to receptors, for example antibodies which bind to Her2receptors as disclosed in International Patent Publications Nos.WO96/32480, WO96/40789 and WO97/04801, which are incorporated herein byreference.

FIG. 4 shows an exemplary structure of a composition of the inventionthat includes a targeting moiety connected to the nitrogen at the 9position of the purine via a connector. The linker may be any generallylinear chain which is of sufficient length permit the targeting moietyand the purine scaffold molecule to interact with their particulartargets when associated together, or may be a linker adapted forcontrolled cleavage once targeting has been accomplished. The linker maybe a C₄ to C₂₀ hydrocarbon chain, or may include intermediateheteroatoms such as O or N. Commonly, the linker is coupled to N-9 ofthe purine scaffold using the same synthetic mechanisms for addingsubstituents to the N-9 position. Terminal functional groups, such asamino or carboxyl groups, on the linker are used to form a bond withreactive sites on the selected targeting moiety.

In lieu of a targeting moiety, the compounds of the invention mayinclude a labeling moiety attached via a connector to the N9 position.Examples of labeling moieties include without limitation biotin. As inthe case of a targeting moiety, the connector is not critical instructure, and need only be of sufficient length so that the labelingmoiety does not interfere with the interaction of the purine scaffoldportion of the molecule with Hsp90.

Use of the Compositions of the Inventions

Because of their ability to bring about the degradation of proteinswhich are essential to cellular function, and hence to retard growthand/or promote cell death, the hsp90-binding compounds of the invention,with or without a targeting moiety, can be used in the therapeutictreatment of a variety of disease conditions. A suitable therapeutic isone which degrades a kinase or protein that is found in enhanced amountsor is mutated in disease-associated cells, or on which the viability ofsuch cells depends. The general role of HSP90 proteins in maintainingmalignancy in most cancer cells points to the importance of this targetin the development of anticancer agents. Thus, the therapeutic smallmolecules of the invention provide a novel modality for the treatment ofall cancers that require or are facilitated by an HSP90 protein. Forexample, the Compositions of the invention can be used in the treatmentof a variety of forms of cancer, particularly those that overexpressHer2 or mutated or wild type steroid receptors, or that lack functionalRB protein. Such cancers may include but are not limited to breastcancer, small cell lung cancer, amyelocytic leukemia, vulvar cancer,non-small cell lung cancer, colon cancer, neuroblastoma and prostatecancer. In addition, the compositions of the invention can be used inthe treatment of other diseases by targeting proteins associated withpathogenesis for selective degradation. Examples of such targetableproteins include antigens associated with autoimmune diseases andpathogenic proteins associated with Alzheimer's disease.

The compositions of the invention exhibit the ability to degradespecific kinases and signaling proteins. Furthermore, selectivity fortransformed versus normal cells can be observed, as reflected in Table3. For example, compound example 1 (FIG. 3B) shows very high selectivityfor tumor cells as opposed to normal heart, brain or lung tissue. Theprecise mechanism for this selectivity is not known, although it isbelieved to arise from a higher affinity for tumor hsp90 as opposed tonormal cells hsp90.

The compositions of the invention are administered to subjects,including human patients, in need of treatment, in an amount effectiveto bring about the desired therapeutic result. A suitable administrationroute is intravenous administration, which is now commonly employed inchemotherapy. In addition, because the compositions of the inventionsare small soluble molecules, they are suitable for oral administration.The ability to use an oral route of administration is particularlydesirable where it may be necessary to provide treatment of a frequent,for example a daily schedule. The amount of any given composition to beadministered, and the appropriate schedule for administration aredetermined using toxicity tests and clinical trials of standard design,and will represent the conclusion drawn from a risk benefit analysis.Such analyses are routinely performed by persons skilled in the art, anddo not involve undue experimentation.

Due to the higher affinity these agents manifest towards cancer cellsand their preferential tumor retention profile, these agents are usefulas tumor imaging agents. They may also be used to monitor the responseof tumors to Hsp90-targeted therapy. The compounds of FIGS. 3F and G(Examples 8 and 9) were iodinated with ¹³¹I using chloamine T andtributlytin precursors. The radioligands were purified by C18 RP HPLC.The saturation binding of these agents to CWR22-rv1 prostate cancercells was undertaken. Animal biodistribution studies and MicroPETimaging were also performed in nude mice with CWR22 transplantabletumors using ¹²⁴I-labeled compounds. These studies showed the uptake ofthe radiolabeled compounds, and the ability to use these compounds inradioimaging to provide an image of a tumor.

Other options for radiolabeling include ¹⁸F which can be used inpositron emission tomography (PET). ¹²³I-labeled compounds can be usedin single photon emission computed tomography (SPECT), and ¹²⁵I-labeledcompounds can be used in surgical gamma probe detection.

The compositions of the invention also have utility to enhance thesensitivity of tumors to other forms of therapy, such as radiation andchemotherapy. This utility can be applied in the context of any type oftumor, but it is particularly relevant in the treatment of gliomas.Given the current therapeutic challenge due to radioresistance andchemoresistance explaining the poor prognosis (median survival of 12months) in GBM, identification of agents that may both sensitize gliomasto radiation and further act as treatments in inhibiting the growth ofthese tumors is necessary. Multipathway-targeted therapy using singleagents that target multiple pathways, including HDAC and Hsp90inhibitors hold promise for improved radiation therapy efficacy and,ultimately, improved patient outcome. Because radiotherapy remains aprimary treatment modality for gliomas, the ability to enhance gliomacell radiosensitivity should provide a therapeutic advantage. Previousstudies using 17AAG and 17DMAG have suggested that Hsp90 is a clinicallyrelevant target for the radiosensitization of a wide variety of tumors(Russell et al, Clinical Cancer Research 9: 3749-3755, 2003; Bull et al,Clinical Cancer Research

10: 8077-8084, 2004). However, whereas in vitro studies have indicatedthat these Hsp90 inhibitors enhance glioma cell radiosensitivity, 17AAGand 17DMAG do not penetrate the blood brain barrier and thus do notappear applicable to brain tumor therapy.

Compound 9 has been shown to have the ability to cross the blood brainbarrier and therefore is suitable for combination with radiotherapy as anovel form of brain tumor treatment. Initial studies based on theclonogenic survival assay indicate that Compound 9 enhances the in vitroradiosensitivity of two human glioma cell lines (U251 and U87) with doseenhancement factors of 1.4-1.6, a degree of radiosensitization similarto that previously shown for 17AAG and 17DMAG. Cells were exposed to 200nM or 400 nM Compound 8 or 9 for 16 h, irradiated with graded doses of Xrays, rinsed and fed with fresh growth media. Colony forming efficiencywas determined 10-12 days later and survival curves generated afternormalizing for cell killing by Compound 9 alone. The results aresummarized in FIG. 11A (Compound 9) and B (Compound 8). The survivingfractions after Compound 9 treatment only were 0.86 and 0.68 for U251and U87 cells, respectively.

Experimental Section

The following procedures and experiments were performed, and areprovided here to further demonstrate the invention.

Hsp90 Competition Assay.

Fluorescence polarization measurements were performed on an Analyst ADinstrument (Molecular Devices, Sunnyvale, Calif.). Measurements weretaken in black 96-well microtiter plates (Corning #3650). The assaybuffer (HFB) contained 20 mM HEPES (K) pH 7.3, 50 mM KCl, 5 mM MgCl₂, 20mM Na₂MoO₄, 0.01% NP40. Before each use, 0.1 mg/mL bovine gamma globulin(BGG) (Panvera Corporation, Madison, Wis.) and 2 mM DTT (Fisher Biotech,Fair Lawn, N.J.) were freshly added. GM-BODIPY was synthesized aspreviously reported^(23a) and was dissolved in DMSO to form 10 μMsolutions. Recombinant Hsp90α was purchased from Stressgen Bioreagents(cat. No. SPP-776), (Victoria, Canada). Cell lysates were preparedrupturing cellular membranes by freezing at −70° C. and dissolving thecellular extract in HFB with added protease and phosphotase inhibitors.Organs were harvested from a healthy mouse and homogenized in HFB.Saturation curves were recorded in which GM-BODIPY (5 nM) was treatedwith increasing amounts of cellular lysates. The amount of lysate thatresulted in polarization (mP) readings corresponding to 20 nMrecombinant Hsp90α was chosen for the competition study. For thecompetition studies, each 96-well contained 5 nM fluorescent GM,cellular lysate (amounts as determined above and normalized to totalHsp90 as determined by Western blot analysis using as standard Hsp90purified from HeLa cells (Stressgen#SPP-770) and tested inhibitor(initial stock in DMSO) in a final volume of 100 μL. The plate was lefton a shaker at 4° C. for 7 h and the FP values in mP were recorded. EC₅₀values were determined as the competitor concentrations at which 50% ofthe fluorescent GM was displaced.

Cell Culture.

The human breast cancer cell lines SKBr3 and MDA-MB-468 were a gift fromDr. Neal Rosen (MSKCC). Cells were maintained in 1:1 mixture of DME:F12supplemented with 2 mM glutamine, 50 units/mL penicillin, 50 units/mLstreptomycin and 10% heat inactivated fetal bovine serum (GeminiBioproducts#100-10b) and incubated at 37° C., 5% CO₂. Growth assays.Growth inhibition studies were performed using the sulforhodamine Bassay as previously described.²⁹ In summary, experimental cultures wereplated in microtiter plates (Nunc#167008). One column of wells was leftwithout cells to serve as the blank control. Cells were allowed toattach overnight. The following day, growth medium having either drug orDMSO at twice the desired initial concentration was added to the platein triplicate and was serially diluted at a 1:1 ratio in the microtiterplate. After 72 h of growth, the cell number in treated versus controlwells was estimated after treatment with 50% trichloroacetic acid andstaining with 0.4% sulforhodamine B in 1% acetic acid. The IC₅₀ wascalculated as the drug concentration that inhibits cell growth by 50%compared with control growth. Normal human renal proximal tubularepithelial (RPTEC) cells were purchased pre-seeded in 96-well plates(Clonetics, CC-3190). Upon receipt, cells were placed in a humidifiedincubator at 37° C., 5% CO₂ and allowed to equilibrate for 3 h. Mediawas removed by suction and replaced with fresh media provided by themanufacturer. Cells were then treated with either drugs or DMSO for 72 hand the IC₅₀ values were determined as described above.

Her2 Assay.

SKBr3 cells were plated in black, clear-bottom microtiter plates(Corning#3603) at 3,000 cells/well in growth medium (100 μl) and allowedto attach for 24 h at 37° C. and 5% CO2. Growth medium (100 μl) withdrug or vehicle (DMSO) was carefully added to the wells, and the plateswere placed at 37° C. and 5% CO₂. Following 24 h incubation with drugs,wells were washed with ice-cold Tris buffer saline (TBS) containing 0.1%Tween 20 (TBST) (200 μl). A house vacuum source attached to aneight-channel aspirator was used to remove the liquid from the plates.Further, methanol (100 μl at −20° C.) was added to each well, and theplate was placed at 4° C. for 10 min. Methanol was removed by washingwith TBST (2×200 μl). After further incubation at RT for 2 h withSuperBlockR (Pierce 37535) (200 μl), anti-Her-2 (c-erbB-2) antibody(Zymed Laboratories#28-004) (100 μl, 1:200 in SuperBlockR) was placed ineach well. The plate was incubated overnight at 4° C. For control wells,1:200 dilution of a normal rabbit IgG (Santa Cruz#SC-2027) inSuperBlockR was used. Each well was washed with TBST (2×200 μl) andincubated at RT for 2 h with an anti-rabbit HRP-linked antibody (Sigma,A-0545) (100 μl, 1:2000 in SuperBlockR). Unreacted antibody was removedby washing with TBST (3×200 μl), and the ECLTM Western blotting reagent(Amersham #RPN2106) (100 μL) was added. The plate was immediately readin an Analyst AD plate reader (Molecular Devices). Each well was scannedfor 0.1 s. Readings from wells containing only control IgG and thecorresponding HRP-linked secondary antibody were set as background anddeducted from all measured values.

Luminescence readings resulted from drug-treated cells versus untreatedcells (vehicle treated) were quantified and plotted against drugconcentration to give the EC₅₀ values as the concentration of drug thatcaused 50% decrease in luminescence.

Anti-Mitotic Assay.

Black, clear-bottom microtiter 96-well plates (Corning Costar#3603) wereused to accommodate experimental cultures. MDA-MB-468 cells were seededin each well at 8,000 cells per well in growth medium (100 μL), andallowed to attach overnight at 37° C. and 5% CO₂. Growth medium (100 μL)with drug or vehicle (DMSO) was gently added to the wells, and theplates were incubated at 37° C. and 5% CO₂ for 24 h. Wells were washedwith ice-cold TBST (2×200 μL). A house vacuum source attached to aneight-channel aspirator was used to remove the liquid from the 96-wellplates. Ice-cold methanol (100 μL) was added to each well, and the platewas placed at 4° C. for 5 min. Methanol was removed by suction andplates were washed with ice-cold TBST (2×200 μL). Plates were furtherincubated with SuperBlock® blocking buffer (Pierce #37535) (200 μL) for2 h at RT. The Tg-3 antibody (gift of Dr. Davies, Albert EinsteinCollege of Medicine) diluted 1:200 in SuperBlock® was placed in eachwell (100 μL) except the control column that was treated with controlantibody (Mouse IgM, NeoMarkers, NC-1030-P). After 72 h, wells werewashed with ice-cold TBST (2×200 μL). The secondary antibody (GoatAnti-Mouse IgM, SouthernBiotech #1020-05) was placed in each well at1:2000 dilution in SuperBlock®, and incubated on a shaker at RT for 2 h.Un-reacted antibody was removed by washing the plates with ice-cold TBST(3×200 μL) for 5 min on a shaker. The ECLTM Western Blotting DetectionReagents 1 and 2 in 1:1 mix (100 μL) was placed in each well and theplates were read immediately in an Analyst AD plate reader (MolecularDevices). Luminescence readings were imported into SOFTmax PROR 4.3.1.Anti-mitotic activity was defined as a concentration dependent increasein luminescence readings in compound-treated wells as compared to DMSOonly treated wells.

General Chemical Procedures.

All commercial chemicals and solvents are reagent grade and were usedwithout further purification. The identity and purity of each productwas characterized by MS, HPLC, TLC, IR and NMR. ¹H NMR/¹³C NMR spectrawere recorded on a Bruker 400 MHz instrument. Low-resolution massspectra (MS) were recorded in the positive ion mode under electron-sprayionization (ESI). High performance liquid chromatography analyses wereperformed on a Waters 2996 instrument with a photodiode array detector(read at 265 nm) and a reverse-phase column (Higgins; HAISIL HL C18 5μm) (method (a)) and additionally, a Waters 2695 Separation Module witha Waters 996 photodiode array detector and a Waters micromass ZQ and areverse-phase column (Varian; Microsorb 100-5 C18 150×2) (methods (b)and (c)). Method (a): 0.1% TFA in water-acetonitrile in the indicatedratio; method (b): 0.05% TFA in water-0.04% TFA in acetonitrile; method(c): 0.05% TFA in water-0.04% TFA in acetonitrile gradient (35%acetonitrile over 18 min, 35-95% acetonitrile over 6 min, 95%acetonitrile over 9 min). Infrared spectra (IR) were obtained on aPerkin-Elmer FT-IR model 1600 spectrometer. Characterization data forpreviously unknown compounds were determined from a single run withisolated yields. Reactions were monitored by thin-layer chromatographyon 0.25-mm silica gel plates and visualized with UV light. Columnchromatography was performed using silica gel (Fisher 170-400 mesh) oralumina (Fisher 60-325 mesh). Oxidation reactions with OXONE® werecarried out in the presence of the Fisher alumina (A540; 80-200 mesh).Analytical thin-layer chromatography (TLC) was performed on E. Merckprecoated silica gel 60 F254. Waters Sep-PakR Vac 6 cc (500 mg) C18cartridges were used for the purification of compounds 16. All reactionswere conducted under inert atmosphere except of those in aqueous media.

3,4,5-Trimethoxy-benzenethiol (6).³⁰

To 3,4,5-trimethoxyaniline (2 g, 10.9 mmol) at 0° C. were added aconcentrated solution of HCl (3 mL, 0.27 mL/mmol) and H₂O (7.7 mL)followed by NaNO₂ (932 mg, 13.1 mmol). The resulting solution was pouredover potassium ethyl xanthogenate (5.35 g, 32.7 mmol) in H2O (6.2 mL)and stirred at 50° C. for 40 min. The reaction mixture was brought toroom temperature, diluted with EtOAc (80 mL) and washed with 10% NaOH,followed by H₂O until the pH reached 7. The organic fraction was driedover Na₂SO₄ and the solvent evaporated under high vacuum. The residuewas purified by column chromatography on silica gel (CH₂Cl₂) to furnishthe xanthogenate intermediate (1.82 g, 58% yield). This was taken up inanhydrous THF (30 mL). To the resulting solution, LiAlH₄ (1 g, 25 mmol)was slowly added and the mixture was stirred for 1 h at refluxtemperature. Following cooling to room temperature, the reaction wasquenched with ice cold water (50 mL) and 10% H₂SO₄ (5 mL) and extractedwith CHCl₃. The organic phase was dried over Na₂SO₄ and

evaporated to give the desired thiophenol (1.18 g, 93% yield). ¹H NMR(CDCl₃) δ 6.53 (s, 2H), 3.84 (s, 6H, OCH3), 3.82 (s, 3H, OCH3), 3.46 (s,1H, SH).

Procedures for the Formation of 8-Arylsulfanyladenine Derivatives:

Scheme 1, synthetic step (b). 8-Mercaptoadenine (7) (50.2 mg, 0.30mmol), neocuproine hydrate (6.8 mg, 0.03 mmol), CuI (5.7 mg, 0.03 mmol),NaO-t-Bu (57.6 mg, 0.6 mmol), the corresponding aryl iodide (0.90 mmol)and anhydrous DMF (2 mL) were charged in a nitrogen box. The reactionvessels were sealed with Teflon tape, placed in an oil bath (110° C.)and magnetically stirred for 24 h. The reaction mixture was then cooledto room temperature and DMF was removed in vacuo. The crude was purifiedby silica gel flash chromatography eluting with a gradient ofCHCl₃:NH₄OH at 10:0.5 to CHCl₃:MeOH:NH₄OH at 10:1:0.5 to afford thedesired product.

8-(2,4,5-Trichloro-phenylsulfanyl)adenine

Use K₂CO₃ as base. Yield, 56%. ¹H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H),8.00 (s, 1H), 7.60 (s, 1H), 7.37 (s, 2H); ¹³C NMR (100 MHz, DMSO-d6) δ132.5, 132.3, 132.2, 131.4, 131.2, 130.8; MS m/z 345.9 (M+H)+. HPLC: (a)99.9% (65% water-35% acetonitrile); (b) 99.4%.

Scheme 1, Synthetic Step (d):

A mixture of 8-arylsulfanyl adenine 10 (100 μmol), Cs2CO3 (100 μmol),pent-4-ynyl 4-methylbenzenesulfonate (120 μmol) in DMF (1.3 mL) undernitrogen protection was heated at 80° C. for 30 min. Following solventremoval, the crude was purified by preparatory TLC with CHCl₃:MeOH:NH4OHat 10:1:0.5 or CHCl₃:MeOH:AcOH at 10:1:0.5 to provide the corresponding9-alkyl-8-arylsulfanyladenine derivatives 11.

9-(Pent-4-ynyl)-8-(2,4,5-trichloro-phenylsulfanyl)adenine (11u)

Yield, 46%. ¹H NMR (400 MHz, CDCl3/MeOD-d4) δ 8.26 (s, 1H), 7.63 (s,1H), 7.50 (s, 1H), 4.38-4.35 (t, J=7.3 Hz, 2H), 2.32-2.28 (m, 2H),2.09-2.02 (m, 3H); ¹³C NMR (100 MHz, CDCl3/MeOD-d4) δ 154.4, 152.7,150.9, 143.8, 134.1, 133.7, 133.4, 131.9, 131.3, 129.4, 81.8, 69.4,42.9, 28.1, 15.7; MS m/z 411.9 (M+H)+. HPLC: (a) 98.5% (75% water-25%acetonitrile); (b) 97.1%.

9-(Pent-4-ynyl)-8-(6-bromo-benzo[1,3]dioxol-5-ylsulfanyl)adenine

Yield, 48%. ¹H NMR (400 MHz, CDCl3/MeOD-d4) δ 8.22 (s, 1H), 7.17 (s,1H), 7.00 (s, 1H), 6.06 (s, 2H), 4.35-4.31 (t, J=7.26 Hz, 2H), 4.12 (s,2H), 2.33-2.30 (m, 2H), 2.08-2.05 (m, 3H). ¹³C NMR (100 MHz,CDCl3/MeODd4) δ 150.9, 149.7, 148.0, 146.9, 121.3, 119.1, 113.8, 113.4,102.4, 81.9, 69.3, 42.7, 28.0, 15.6; MS m/z 432.0 (M+H)+. HPLC: (a)98.7% (75% water-25% acetonitrile); (b) 98.9%.

9-(2-Isopropoxy-ethyl)-8-(2,4-dichloro-5-methoxy-benzenesulfanyl)adenine

Following the general method for the preparation of 12d, 11e3 afforded12c. Yield, 53%. ¹H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H, H-2), 7.44 (s,1H), 7.09 (s, 1H), 5.55 (bs, 2H, NH2), 4.48 (t, J=5.6 Hz, 2H, NCH2),3.81 (s, 3H, OCH3), 3.74 (t, J=5.6 Hz, 2H), 3.49 (d, J=6.1 Hz, 1H, CH),1.04 (d, J=6.1 Hz, 6H, CH3); 13C NMR (100 MHz, CDCl3) δ 154.1 (C-2),153, 147, 131.0, 130.2, 126.9, 123.6, 116.0, 72.3 (CH), 65.6, 56.5(OCH3), 44.1 (NCH2), 21.8 (CH2); MS (EIS) m/z 428.0 (M+1)+. HPLC: (a)90.2% (70% water-30% acetonitrile); (c) 91.0%.

Method for the Fluorination of the Adenine Moiety at C2:

2-Fluoro-9-butyl-8-(2-chloro-3,4,5-trimethoxy-phenylsulfanyl)adenine(21c). To a cooled solution (0° C.) of 18c (11.3 mg, 0.02 mmol) inHF/pyridine (18 μL, 0.7 mL/mmol) NaNO2 (2.2 mg, 0.03 mmol) was slowlyadded. The resulting mixture was stirred at room temperature for 1 h andthen quenched by stirring for 1 h with 14 mg of CaCO3 in CH2Cl2 (75 μL).The crude was taken up in CH2Cl2, washed with water and dried overanhydrous Na2SO4. Following solvent removal, the residue was purified ona preparative silica gel plate (CHCl3:Hexanes:EtOAc:i-PrOH at 2:2:1:0.1)to afford 21c (1.9 mg, 17% yield). IR (film) ν_(max) 3318-2953, 1657,1604, 1583, 1479, 1385, 1111, 1015; ¹H NMR (400 MHz, CDCl3) δ 6.72 (s,1H), 5.83 (bs, 2H, NH2), 4.18 (t, J=7.5 Hz, 2H, NCH2), 3.92 (s, 3H,OCH3), 3.89 (s, 3H, CH3), 3.74 (s, 3H, CH3), 1.72 (m, 2H), 1.32 (m, 2H),0.92 (t, J=7.4 Hz, 3H, CH3); ¹³C NMR (100 MHz, CDCl3) δ 160.1, 158.0,156.1, 152.5, 150.8, 143.9, 124.6, 111.2, 61.2 and 56.3 (OCH3), 43.9(NCH2), 31.7, 29.7, 19.7 (CH3); MS (EIS) m/z 442.2 (M+1)+. HPLC: (a)95.9% (60% water-40% acetonitrile); (c) 98.0%.

8-(6-Bromo-benzo[1,3]dioxol-5-ylsulfanyl)adenine

8-Mercaptoadenine (602 mg, 3.6 mmol), neocuproine hydrate (81 mg, 0.36mmol), CuI (69 mg, 0.36 mmol), NaO-t-Bu (692 mg, 7.2 mmol),5-bromo-6-iodo-benzo[1,3]dioxole (3.53 g, 10.8 mmol) and anhydrous DMF(24 mL) were charged in a nitrogen box. The vessel was sealed withTeflon tape, placed in an oil bath (110° C.) and magnetically stirredfor 24 h. The solvent was removed under high vacuum and the crudepurified by column chromatography on silica gel (EtOAc:CH2Cl2:MeOH at2:2:1) to provide the product (1.29 g, 97%). ¹H NMR (400 MHz,acetone-d6) 8.07 (s, 1H), 7.28 (s, 1H), 7.15 (s, 1H), 7.08 (bs, 2H),6.13 (s, 2H); MS m/z 366.0 (M+H)+.

8-(6-Iodo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(pent-4-ynyl)adenine

A solution of 7 (40 mg, 96.8 mol), Cs2CO3 (31.5 mg, 96.8 mol) andpent-4-ynyl tosylate (28 mg, 114 mol) in anhydrous DMF (1 mL) wasstirred at 80° C. for 30 min. The solvent was removed under high vacuumand the crude purified by preparatory thin layer chromatography to givethe desired product (25 mg, 53.9%): 1H NMR (400 MHz, CDCl3/methanol-d4)8.23 (s, 1H), 7.38 (s, 1H), 7.04 (s, 1H), 6.05 (s, 2H), 4.32 (t, J=7.3Hz, 2H), 2.33-2.31 (m, 2H), 2.12-2.04 (m, 3H); 13C NMR (100 MHz,CDCl3/methanol-d4) 151.1, 149.6, 149.2, 147.5, 125.7, 119.4, 113.6,102.4, 93.8, 82.1, 69.4, 42.8, 28.1, 15.8; MS m/z 480.0 (M+H)+. HPLC:(a) 98.5% (65% water-35% acetonitrile); (b) 97.7% (35% to 95%acetonitrile).

3-(tert-Butoxycarbonyl-isopropyl-amino)-propyl tosylate: A solution of3-bromo-1-propanol (5 g, 0.036 mol) in isopropylamine (9 mL, 0.11 mol)was heated overnight at 50° C. with stirring. Solvent was removed undervacuum to give the product, 3-isopropyl-amino-propanol as a white solid.To this were added di-tert-butyl dicarbonate (10 g, 0.05 mol) andtriethylamine (11 mL, 0.08 mol) and the resulting solution stirred atroom temperature overnight. Following solvent removal, the crude waspurified by column chromatography on silica gel (CH2Cl2, thenCH2Cl2:acetone at 3:1) to provide the3-(tert-butoxycarbonyl-isopropyl-amino)-propanol (5.8 g, 75%). 1H NMR(400 MHz, CDCl3) 3.93 (bs, 1H), 3.58 (m, 2H), 3.33 (m, 2H), 1.67 (m,2H), 1.48 (s, 9H), 1.16 (d, J=6.9 Hz, 6H); MS m/z 218.1 (M+H)+. Asolution of 3-(tert-butoxycarbonyl-isopropyl-amino)-propanol (3.5 g,0.016 mol), p-toluenesulfonyl chloride (3.7 g, 0.019 mol) and pyridine(1.6 mL, 0.019 mol) in CH2Cl2 (50 mL) was stirred overnight at roomtemparature. Following solvent removal, the product (2.3 g, 40%) wasisolated by column chromatography on silica gel (hexanes:CH2Cl2:EtOAc at5:4:1). 1H NMR (400 MHz, CDCl3) 7.79 (d, J=8.2 Hz, 2H,), 7.35 (d, J=8.2Hz, 2H,), 4.06-4.03 (m, 3H), 3.09 (t, J=6.5 Hz, 2H), 2.45 (s, 3H),1.91-1.87 (m, 2H), 1.42 (s, 9H), 1.08 (d, J=6.7 Hz, 6H); MS m/z 372.2(M+H)+.

8-(6-Iodo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(3-isopropylamino-propyl)adenine

A solution of 7 (125 mg, 303 mol),3-(tert-butoxycarbonyl-isopropyl-amino)-propyl tosylate (269 mg, 726mol), Cs2CO3 (99 mg, 303 mol) in anhydrous DMF (2 mL) was stirred at 80°C. for 24 h. The solvent was removed under vacuum and the crude purifiedby preparatory thin layer chromatography on silica gel (CHCl3:MeOH:NH4OHat 10:1:0.5) to afford the 9N-alkylated compound. This was placed in TFA(1 mL) at 0° C. for 1.5 h to remove the Boc protecting group and yield11 (30 mg, 19.3% yield): 1H NMR (400 MHz, CDCl3) 8.31 (s, 1H), 7.29 (s,1H), 6.88 (s, 1H), 6.10 (bs, 2H), 5.96 (s, 2H), 4.29 (t, J=7.0 Hz, 2H),2.75-2.69 (m, 1H), 2.58 (t, J=6.8 Hz, 2H), 2.02-1.95 (m, 2H), 1.03 (d,J=6.2 Hz, 6H); 13C NMR (100 MHz, CDCl3) 154.6, 152.9, 151.6, 149.2,148.9, 146.2, 127.9, 120.1, 119.2, 112.2, 102.2, 91.1, 48.7, 43.9, 41.7,30.3, 22.9; MS m/z 513.2 (M+H)+. HPLC: (a) 98.9% (65% water-35%acetonitrile); (b) 95.0% (20% to 40% acetonitrile).

8-(6-Bromo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(3-isopropylamino-propyl)adenine

A solution of 8-(6-Bromo-benzo[1,3]dioxol-5-ylsulfanyl)adenine (513 mg,1.4 mmol), PPh3 (808 mg, 3.08 mmol), 3-bromo-1-propanol (253 mg, 165 L,1.82 mmol), DBAD (1612 mg, 7 mmol) in toluene (43.8 mL) and CH2Cl2(8.75, mL) was stirred at room temperature for 20 min. The reactionmixture was loaded to a silica gel column (CHCl₃ thenCHCl₃:EtOAc:hexanes:i-Propanol at 4:4:2:1) to provide the 9N-alkylatedcompound,8-(6-bromo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(3-bromo-propyl)adenine)(142.6 mg, 21% yield). A solution of this product (142.6 mg, 0.29 mmol)in 1,4-dioxane (12 mL) and i-propylamine (3 mL) was stirred at 100° C.for 2.5 h. The solvent was removed under vacuum and the crude purifiedby preparatory thin layer chromatography on silica gel (CHCl3:MeOH:NH4OHat 10:1:0.5 then EtOAc:CH2Cl2:MeOH at 2:2:1) to afford 12 (51 mg, 8%yield). 1H NMR (400 MHz, CDCl3) 8.30 (s, 1H), 7.04 (s, 1H), 6.81 (s,1H), 6.48 (bs, 2H), 5.94 (s, 2H), 4.29 (t, J=7.0 Hz, 2H,), 2.74-2.68 (m,1H), 2.57 (t, J=6.8 Hz, 2H), 2.02-1.95 (m, 2H), 1.02 (d, J=6.0 Hz, 6H).13C NMR (100 MHz, CDCl3) 154.8, 152.9, 151.5, 148.8, 148.0, 145.2,123.8, 120.0, 116.7, 113.2, 112.2, 102.3, 48.6, 43.8, 41.7, 30.2, 22.8;MS m/z 465.0 (M+H)+. HPLC: (a) 99.1% (65% water-35% acetonitrile); (b)98.0% (20% to 40% acetonitrile).

8-Benzo[1,3]dioxol-5-ylmethyl-2-fluoroadenine

To a cooled (0° C.) solution of 16 (1.48 g, 5.2 mmol) in HF/pyridine(3.64 mL), NaNO2 (0.47 g, 6.76 mmol) was slowly added. The reaction wasbrought to room temperature, and further stirred for 1 h. Followingdilution with CH₂Cl₂ (38 mL), the excess HF was quenched by stirring foran additional 1 h with CaCO3 (0.95 g) and water (5 mL). The mixture wasdried in vacuo and subsequently purified by silica gel columnchromatography (CHCl3:MeOH:NH4OH at 5:1:0.5) to yield 17 (0.9 g, 60%yield). 1H NMR (400 MHz, DMSO-d6) 7.59 (bs, 2H), 6.94-6.90 (m, 3H), 6.81(d, J=8.0 Hz, 1H), 6.03 (s, 2H), 4.06 (s, 2H); MS m/z 288.0 (M+H)+.

2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)adenine

A solution of 8-Benzo[1,3]dioxol-5-ylmethyl-2-fluoroadenine (50 mg, 0.17mmol), NIS (94 mg, 0.4 mmol), TFA (20 mg, 13.4 L, 0.17 mmol) in CH2Cl2(200 L) was stirred at room temperature overnight. After solventremoval, the desired product 18 (6 mg, 8.5%) was purified by silica gelcolumn chromatography (CHCl3:EtOAc at 9:1 to 4:6). 1H NMR (400 MHz,DMSO-d6) 7.6 (bs, 2H), 7.38 (s, 1H), 6.95 (s, 1H), 6.03 (s, 2H), 4.12(s, 2H); MS m/z 414.1 (M+H)+.

2-Fluoro-8-(6-bromo-benzo[1,3]dioxol-5-ylmethyl)adenine

A solution of 8-Benzo[1,3]dioxol-5-ylmethyl-2-fluoroadenine (45 mg,0.157 mmol), NBS (56 mg, 0.314 mmol) in DMF (0.5 mL) was stirred at roomtemperature for 1.5 h. Following solvent removal, product (20 mg, 34.8%)was collected through silica gel column purification (CHCl3:EtOAc at 9:1to 4:6). 1H NMR (400 MHz, acetone-d6) 7.51 (bs, 2H), 7.21 (s, 1H), 6.98(s, 1H), 6.06 (s, 2H), 4.13 (s, 2H); MS m/z 366.0 (M+H)+.

2-Fluoro-8-(6-chloro-benzo[1,3]dioxol-5-ylmethyl)adenine

A solution of 8-Benzo[1,3]dioxol-5-ylmethyl-2-fluoroadenine (20 mg, 0.07mmol), NCS (35.6 mg, 0.27 mmol) in anhydrous DMF (0.4 mL) was stirred atroom temperature for 2.5 h. Following solvent removal, the product (11mg, 48.8%) was collected through silica gel column purification(CHCl3:EtOAc at 9:1 to 5:5). 1H NMR (400 MHz, DMSO-d6) 7.40 (bs, 2H),6.97 (s, 1H), 6.89 (s, 2H), 5.97 (s, 2H), 4.04 (s, 2H); MS m/z 322.1(M+H)+.

2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)-9-(pent-4-ynyl)adenine

A solution of 2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)adenine (6mg, 0.0145 mmol), Cs2CO3 (5 mg, 0.0145 mmol) and pent-4-ynyl tosylate(4.5 mg, 0.189 mmol) in anhydrous DMF (200 L) was stirred at 60° C. for1.5 h. Following solvent removal, product (5.9 mg, 84.9%) was collectedthrough silica gel column purification (EtOAc:hexanes:CHCl3:i-PrOH at10:20:20:1). 1H NMR (400 MHz, CDCl3) 7.29 (s, 1H), 6.59 (s, 1H), 5.94(s, 2H), 5.83 (bs, 2H), 4.26 (s, 2H), 4.11 (t, J=7.4 Hz, 2H), 2.26-2.19(m, 2H), 2.00 (t, J=2.5 Hz, 1H), 1.98-1.94 (m, 2H); 13C NMR (100 MHz,CDCl3) 150.9, 148.9, 147.8, 131.5, 118.8, 109.4, 101.9, 88.1, 82.3,69.9, 42.3, 39.2, 28.2, 15.9; MS m/z 480.0 (M+H)+. HPLC: (a) 95.5% (60%water-40% acetonitrile); (b) 95.0% (35% to 55% acetonitrile).

2-Fluoro-8-(6-bromo-benzo[1,3]dioxol-5-ylmethyl)-9-(pent-4-ynyl)adenine

A solution of 2-Fluoro-8-(6-bromo-benzo[1,3]dioxol-5-ylmethyl)adenine(20 mg, 55 mol), Cs2CO3 (18 mg, 55 mol) and pent-4-ynyl tosylate (17 mg,72 mol) in anhydrous DMF (138 L) was stirred at 60° C. for 2 h.Following solvent removal, the product (13 mg, 54.7%) was collectedthrough silica gel column purification (EtOAc:hexanes:CHCl3:i-PrOH at10:20:20:1). 1H NMR (400 MHz, CDCl3) 7.05 (s, 1H), 6.60 (s, 1H), 6.15(bs, 2H), 5.96 (s, 2H), 4.28 (s, 2H), 4.13 (t, J=7.5 Hz, 2H), 2.25-2.21(m, 2H), 2.00 (t, J=2.6 Hz, 1H), 1.98-1.92 (m, 2H); 13C NMR (100 MHz,CDCl3) 157.6, 156.0, 152.6, 150.2, 147.5, 127.6, 116.4, 114.1, 112.5,109.5, 101.6, 81.9, 69.5, 41.9, 33.7, 27.8, 15.5; MS m/z 432.0 (M+H)+.HPLC: (a) 99.0% (60% water-40% acetonitrile); (b) 98.5% (35% to 55%acetonitrile).

2-Fluoro-8-(6-chloro-benzo[1,3]dioxol-5-ylmethyl)-9-(pent-4-ynyl)adenine

A solution of 2-Fluoro-8-(6-chloro-benzo[1,3]dioxol-5-ylmethyl)adenine(11 mg, 0.034 mmol), Cs2CO3 (11 mg, 0.034 mmol) and pent-4-ynyl tosylate(10.5 mg, 0.044 mmol) in anhydrous DMF (85 L) was stirred at 50° C. for1 h. Following solvent removal, the product (4.2 mg, 31.9%) wascollected through silica gel column purification(EtOAc:hexanes:CHCl3:i-PrOH at 10:20:20:1). 1H NMR (400 MHz, CDCl3) 6.89(s, 1H), 6.61 (s, 1H), 5.98 (bs, 2H), 5.96 (s, 2H), 4.27 (s, 2H), 4.13(t, J=7.5 Hz, 2H), 2.24-2.10 (m, 2H), 2.00-1.91 (m, 3H); 13C NMR (100MHz, CDCl3) 1597, 158.0, 156.3, 150.6, 147.7, 147.2, 126.2, 125.3,110.0, 102.0, 82.3, 69.9, 42.2, 31.4, 28.1, 15.8; MS m/z 388.1 (M+H)+.HPLC: (a) 98.1% (65% water-35% acetonitrile); (b) 97.0% (35% to 45%acetonitrile).

2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)-9-(3-isopropylamino-propyl)adenine

A solution of 2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)adenine(300 mg, 0.726 mmol), Cs2CO3 (285 mg, 0.87 mmol) and 1,3-dibromopropane(370 L, 3.63 mmol) in anhydrous DMF (5 mL) was stirred at 50° C. for 2h. Following solvent removal, product (330 mg, 85%) was collectedthrough silica gel column purification (CHCl3 thenEtOAc:hexanes:CHCl3:i-PrOH at 4:2:4:0.4). MS m/z 534.0 (M+H)+. To thisproduct, i-PrNH2 (10 mL) was added in excess and the resulting solutionstirred at room temperature for 1 h. Excess amine was removed andproduct (230 mg, 75%) collected through silica gel column purification(CHCl3:EtOAc:i-PrOH:NH4OH at 4:4:2:0.3). 1H NMR (400 MHz, CDCl3) 7.29(s, 1H), 6.59 (s, 1H), 5.94 (s, 2H), 5.89 (bs, 2H), 4.25 (s, 2H), 4.11(t, J=7.0 Hz, 2H), 2.73-2.60 (m, 1H), 2.55 (t, J=6.8 Hz, 1H), 1.93-1.86(m, 2H), 1.03-1.02 (d, J=6.0 Hz, 6H); 13C NMR (100 MHz, methanol-d4)160.0, 158.4, 157.2, 152.4, 151.3, 149.4, 148.4, 133.1, 118.7, 110.6,102.4, 88.5, 42.8, 40.1, 38.8, 27.6, 19.4; MS m/z 513.2 (M+H)+. HPLC:(a) 98.5% (60% water-40% acetonitrile); (b) 97.2% (20% to 50%acetonitrile).

2-Fluoro-8-(3,4-dimethoxy-benzyl)adenine

Starting from 2-amino-8-(3,4-dimethoxy-benzyl)adenine (0.66 g, 2.2 mmol)and following the procedure for the synthesis of 17, the desired productwas obtained (0.34 g, 51%). 1H NMR (400 MHz, DMSO-d6) 7.61 (bs, 2H),7.03 (s, 1H), 6.94 (d, J=8.3 Hz, 1H), 6.84 (d, J=8.6 Hz, 1H), 4.07 (s,2H), 3.80 (s, 3H), 3.77 (s, 3H); MS m/z 304.0 (M+H)+.

2-Fluoro-8-(2-iodo-4,5-dimethoxy-benzyl)adenine

A solution of 2-Fluoro-8-(3,4-dimethoxy-benzyl)adenine (50 mg, 0.165mmol), NIS (74 mg, 0.33 mmol), TFA (18.8 mg, 12.7 L, 0.165 mmol) inacetonitrile (120 L) was stirred at room temperature for 24 h. Followingsolvent removal, the product (12 mg, 16.9%) was collected through silicagel column purification (CHCl3:MeOH:AcOH at 80:1:0.5 to 30:1:0.5). MSm/z 430.1 (M+H)+.

2-Fluoro-8-(2-bromo-4,5-dimethoxy-benzyl)adenine (29): A solution of 27(65 mg, 0.226 mmol), NBS (80 mg, 0.45 mmol) in DMF (0.75 mL) was stirredat room temperature for 2.5 h. Following solvent removal, the product(8.2 mg, 53.6%) was collected through silica gel column purification(CHCl3:MeOH:AcOH at 80:1:0.5 to 30:1:0.5). 1H NMR (400 MHz, aceton-d6)7.13 (s, 1H), 7.09 (s, 1H), 6.80 (bs, 2H), 4.26 (s, 2H), 3.84 (s, 3H),3.78 (s, 3H); MS m/z 382.0 (M+H)+.

2-Fluoro-8-(2-chloro-4,5-dimethoxy-benzyl)adenine

A solution of 2-Fluoro-8-(3,4-dimethoxy-benzyl)adenine (40 mg, 0.132mmol), NCS (77.8 mg, 0.58 mmol) in anhydrous DMF (0.7 mL) was stirred atroom temperature for 5.5 h. Following solvent removal, the product (22mg, 49.4%) was collected through silica gel column purification(CHCl3:EtOAc at 8:2 to 4:6). MS m/z 338.0 (M+H)+.

2-Fluoro-8-(2-iodo-4,5-dimethoxy-benzyl)-9-(pent-4-ynyl)adenine

A solution of 2-fluoro-8-(2-iodo-4,5-dimethoxy-benzyl)adenine (12 mg,0.028 mmol), Cs2CO3 (9 mg, 0.028 mmol), pent-4-ynyl tosylate (8.6 mg, 7L, 0.036 mmol) in anhydrous DMF (80 L) was stirred at 50° C. for 1 h.Following solvent removal, the product (13.7 mg, 99%) was collectedthrough silica gel column purification (CHCl3:EtOAc:hexanes:i-PrOH at20:10:20:1). 1H NMR (400 MHz, CDCl3) 7.27 (s, 1H), 6.65 (s, 1H), 5.94(bs, 2H), 4.29 (s, 2H), 4.13 (t, J=7.3 Hz, 2H), 3.87 (s, 3H), 3.73 (s,3H), 2.26-2.22 (m, 2H), 2.00 (t, J=2.6 Hz, 1H), 1.97-1.90 (m, 2H); 13CNMR (100 MHz, CDCl3) 156.5, 153.2, 151.3, 150.0, 149.1, 130.9, 121.9,112.6, 88.5, 82.5, 70.0, 56.4, 56.2, 42.6, 39.2, 28.4, 16.1; MS m/z496.2 (M+H)+. HPLC: (a) 99.9% (60% water-40% acetonitrile); (b) 96.8%(35% to 55% acetonitrile).

2-Fluoro-8-(2-bromo-4,5-dimethoxy-benzyl)-9-(pent-4-ynyl)adenine

A solution of 8-(2-bromo-4,5-dimethoxy-benzyl)-2-fluoroadenine (13 mg,0.034 mmol), Cs2CO3 (11 mg, 0.034 mmol), pent-4-ynyl tosylate (10 mg, 9L, 0.044 mmol) in anhydrous DMF (80 L) was stirred at 60° C. for 30 min.Following solvent removal, the product (8.2 mg, 53.6%) was collectedthrough silica gel column purification (CHCl3:EtOAc:hexanes:i-PrOH at20:10:20:1). 1H NMR (400 MHz, CDCl3) 7.06 (s, 1H), 6.67 (s, 1H), 5.92(bs, 2H), 4.31 (s, 2H), 4.14 (t, J=7.4 Hz, 2H), 3.88 (s, 3H), 3.75 (s,3H), 2.25-2.20 (m, 2H), 1.99 (t, J=2.6 Hz, 1H), 1.96-1.89 (m, 2H); 13CNMR (100 MHz, CDCl3) 160.0, 158.3, 156.7, 153.4, 151.3, 149.4, 127.2,117.3, 115.9, 114.5, 113.2, 82.7, 70.2, 56.62, 56.56, 42.7, 34.3, 30.1,28.5, 16.3; MS ink 447.9 (M+H)+. HPLC: (a) 99.0% (60% water-40%acetonitrile); (b) 98.8% (35% to 55% acetonitrile).

2-Fluoro-8-(2-chloro-4,5-dimethoxy-benzyl)-9-(pent-4-ynyl)adenine

A solution of 8-(2-chloro-4,5-dimethoxy-benzyl)-2-fluoroadenine (22 mg,0.065 mmol), Cs2CO3 (21 mg, 0.065 mmol), pent-4-ynyl tosylate (20 mg,17.3 L, 0.085 mmol) in anhydrous DMF (170 L) was stirred at 50° C. for 2h. Following solvent removal, the product (14 mg, 53.8%) was collectedthrough silica gel column purification (CHCl3:EtOAc:hexanes:i-PrOH at20:10:20:1). 1H NMR (400 MHz, CDCl3) 6.91 (s, 1H), 6.67 (s, 1H), 6.01(bs, 2H), 4.31 (s, 2H), 4.14 (t, J=7.5 Hz, 2H), 3.87 (s, 3H), 3.75 (s,3H), 2.24-2.20 (m, 2H), 2.01-1.99 (m, 1H), 1.97-1.88 (m, 2H); 13C NMR(100 MHz, CDCl3) 1596, 157.9, 156.3, 152.3, 148.9, 146.0, 124.9, 112.7,82.3, 69.9, 56.3, 56.2, 42.2, 31.2, 28.1, 15.9; MS m/z 404.1 (M+H)+.HPLC: (a) 95.1% (65% water-35% acetonitrile); (b) 96.7% (35% to 45%acetonitrile).

Radiolabeling of Examples 8 and 9

Ten microliters of [¹³¹I]—NaI (3 mCi) in 0.1M NaOH is added to a 0.3 mLReactiVial followed by 5 μL of a 5 μg/μL methanol solution of2-fluoro-9-[3-(N—N-tert-butoxycarboxy-2-propylamino)propyl]-8-(4-trimethylstannyl-1,3-benzodioxol-5-yl)methyladenine followed by 10 μL of Chloramine-T (CAT) in acetic acid (0.5mg/mL). The reaction mixture is vortexed and kept at 50° C. for 5minutes. 10 μL 6M HCl is added, the reaction mixture is vortexed andkept at 50° C. for 15 minutes. 6 μL 10M NaOH is added, the reactionmixture is vortexed and injected into a HPLC (Phenomenex Luna C18 column(5 μm, 4.4×250 mm). Both columns were eluted at 1 mL/min with a solventgradient of 0.1% TFA to 0.1% TFA/70% acetonitrile over 15 minutes. ThePU-DZ8 fraction is collected, dried at 50° C. by a stream of nitrogen,reconstituted in saline and sterile filtered to yield ˜90% radiochemicalyield of [¹³¹I]-Compound 8. [¹²⁴I]-Compound 8 and [¹³¹I]-Compound 9 areproduced in an analogous manner.

Binding Studies

CWR22-rv1 prostate cancer cells are grown in RMPI 1640 mediasupplemented with 10% fetal bovine serum at 37° C. The cells are removedfrom the flasks using trypsin and propagated with a 1:6 subcultureratio.

Displacement Binding Studies

Displacement studies are performed with [¹³¹I]-Compound 8 and CWR22-rv1prostate cancer cells. Briefly, triplicate samples of cells are mixedwith <1 nM of radioligand and increasing amounts of a cold competitor (1pM to 1 μM Compound 8 or 9). The solutions are shaken on an orbitalshaker and after 60 minutes the cells are isolated and washed with icecold Tris buffered saline using a Brandel cell harvester. All theisolated cell samples are counted and the specific uptake of[¹³¹I]-Compound 8 determined. These data are plotted against theconcentration of the cold competitor to give sigmoidal displacementcurves. The IC₅₀ values are determined using a one site model and aleast squares curve fitting routine. The displacement binding of[¹³¹I]-Compound 8 is determined in an analogous manner. FIG. 11 showsexample displacement curves for and [¹³¹I]-Compound 8 and [¹³¹I]-Compound 9.

Saturation Binding Studies

Saturation studies are performed with [¹³¹I]-Compound 8 and CWR22-rv1prostate cancer cells. Briefly, triplicate samples of cells are mixedwith increasing amount of 131I-DZ8 either with or without 1 μM unlabeledCompound 9. The solutions are shaken on an orbital shaker and after 60minutes the cells are isolated and washed with ice cold Tris bufferedsaline using a Brandel cell harvester. All the isolated cell samples arecounted and the specific uptake of ¹³¹I-Compound 8 determined. Thesedata are plotted against the concentration of ¹³¹I-Compound to give asaturation binding curve. The Bmax (maximal binding) and Kd (bindingaffinity) are determined by using a least squares curve fitting routine.The saturation binding of [¹³¹I]-Compound 9 is determined in ananalogous manner. FIG. 12 shows example saturation curves for[¹³¹I]-Compound 8 and [¹³¹I]-Compound 9.

Animal Studies

[¹³¹I]-Compound 8 and [¹³¹ I]-Compound 9 biodistribution was studied inan animal model of prostate cancer. CWR22 tumors are grown in athymicmice supplemented with a testosterone pellet (12.5 mg pellet, InnovativeResearch of America, Sarasota, Fla.). Once the tumors are 500 mg in sizethe pellet is removed and the mice castrated.

Two groups of eight mice were injected at 3 days post castration, with10 μCi of [¹³¹I]-Compound 8 or [¹³¹I]-Compound 9. These mice weresacrificed at either 4 or 24 hours post injection and the organs ofinterest removed, weighed and counted in a gamma counter with a standardof 10% of the [¹³¹I]-Compound 8 injected dose. The data were thenexpressed as a % of the injected dose per gram of tissue (% ID/g). FIG.13 shows the tumor/non-tumor uptake ratios for selected organs. Asshown, there is a substantial excess of accumulation of the radiolabeledcompound in tumor as compared to both muscle and blood, and this ratioincreases over time.

In a second study, 14 mice were injected with [¹³¹I]-Compound 9 withincreasing amounts of unlabeled Compound 9. The mice were sacrificed at4 h p.i. and tissue analyzed as described above. While the addition of 2or 18 nanomoles of unlabeled Compound 9 decreased the amount of capturedlabel to some extent, it did not alter the tissue distribution to anysignificant extent.

In a third study a single mouse was injected with [¹²⁴I]-Compound 8 andimaged with a microPET at around 3 and 17 hours post injection. The areaof the tumor was plainly visible in the images obtained, along withresidual activity in the large intestine.

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The invention claimed is:
 1. An acid addition salt of the compound: